Fighting resistance: post-PARP inhibitor treatment strategies in ovarian cancer

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a therapeutic milestone in the management of epithelial ovarian cancer. The concept of ‘synthetic lethality’ is exploited by PARPi in tumors with defects in DNA repair pathways, particularly homologous recombination deficiency. The use of PARPis has been increasing since its approval as maintenance therapy, particularly in the first-line setting. Therefore, resistance to PARPi is an emerging issue in clinical practice. It brings an urgent need to elucidate and identify the mechanisms of PARPi resistance. Ongoing studies address this challenge and investigate potential therapeutic strategies to prevent, overcome, or re-sensitize tumor cells to PARPi. This review aims to summarize the mechanisms of resistance to PARPi, discuss emerging strategies to treat patients post-PARPi progression, and discuss potential biomarkers of resistance.


Introduction
Epithelial ovarian cancer (EOC) continues to represent the second leading cause of gynecologic cancer death worldwide. 1 High-grade serous ovarian cancer (HGSOC) is the most frequent histology, often diagnosed in advanced stages (III/IV). 2 For the past two decades, the standard of care has been debulking surgery and systemic treatment with platinum-based chemotherapy in the first-line setting. Nevertheless, about 70% of patients with advanced disease relapse within the first 3 years. 3 The first relapse is often sensitive to platinum; however, the disease becomes drug resistant over time. Since the potential synergy for targeting PARPi to BRCA1 and BRCA2 mutations was first reported in 2005, the treatment landscape of EOC has changed. 4 Inhibition of PARP is synthetically lethal in EOC with BRCA1/2 mutation and other aberrations leading to homologous recombination deficiency (HRD). 5 The Cancer Genome Atlas has described that ~50% of HGSOC may harbor some form of HRD. In addition, HGSOC is characterized by TP53 mutation, which plays an important role in the cell-cycle regulation. Therefore, HGSOC is susceptible to DNA-damaging drugs, such as platinum agents and PARPi. 6 The integrity of double-stranded DNA is essential to maintaining genomic stability. DNA damage and its repair deficiencies have an essential role in HGSOC oncogenesis and their response to treatment. The DNA damage repair (DDR) system involves a complex molecular network of interconnected signaling pathways, with the main objective being the repair of DNA doublestranded breaks (DSBs) to maintain cell integrity. 7 One of the most critical cell repair tools is homologous recombination (HR). HRD is a functional alteration whereby the DNA is unable to be repaired by HR. Consequently, cells employ alternative DSB repair by nonhomologous end joining (NHEJ), single-strand annealing, or microhomology-mediated end joining (MMEJ) pathways, which are error-prone DNA repair pathways causing high mutation rates. 8 Unrepaired DSBs accumulate genomic aberrations resulting in instability which drives

Fighting resistance: post-PARP inhibitor treatment strategies in ovarian cancer
oncogenesis and can be detected as a 'genomic scar'. These genomic changes are permanent and could represent mutations, insertions/deletions, and rearrangements. However, functional HR repair (HRR) can change over time (e.g. cells may regain the ability to repair DSBs) and drive mechanisms of drug resistance. HRD biomarkers include BRCA1/2 and non-BRCA1/2 HRD gene pathogenic or likely pathogenic variants and have become therapeutic targets, as well as predictive biomarkers. 9 Poly (ADP-ribose) polymerase inhibitors (PARPis) have been approved in the first-line maintenance setting with unprecedented results in patients with EOC, especially in BRCAdeficient tumors. A recent analysis of long-term overall survival (OS) at 7 years in the SOLO-1 phase III trial (NCT01844986) confirmed the benefit of first-line maintenance olaparib in BRCA-mutated EOC. Olaparib compared to placebo led to clinically meaningful improvement in OS in patients with newly diagnosed advanced EOC harboring a BRCA mutation. 10 The median OS was not reached in the olaparib group compared to 75.2 months in the placebo group (HR: 0.55, 95% CI: 0.40-0.76; p = 0.0004). This result is particularly remarkable as 44% crossover was reported, with data maturity of 38.1%. In the PAOLA-1 phase III trial (NCT02477644), no significant benefit in OS was observed with the addition of olaparib to bevacizumab compared to placebo and bevacizumab in first-line maintenance therapy in women with newly diagnosed advanced EOC. However, a significant improvement in median OS and progression-free survival (PFS) was observed in the HRD-positive subgroup. In this subgroup, the 5-year OS rates were 65.5% with olaparib plus bevacizumab versus 48.4% with bevacizumab and placebo (HR: 0.62, 95% CI: 0.45-0.85). 11 The phase III PRIMA trial (NCT02655016) demonstrated benefit of niraparib in first-line maintenance irrespective of BRCA or HRD status. 12 Updated data with 3.5 years follow-up revealed a sustained median PFS of 13.8 months with niraparib versus 8.2 months with placebo in all-comers (HR: 0.66; 95% CI: 0.56-0.79; p < 0.001). 13 Recently, data from the phase III ATHENA-MONO trial (NCT03522246) also demonstrated that firstline maintenance rucaparib significantly improved median PFS in all-comers regardless of HRD status (HR: 0.52; 95% CI: 0.56-0.79; p < 0.0001). 14 Despite impressive outcomes of PARPi, patients can present with de novo or, more frequently, acquired PARPi resistance. Studies of resistance mechanisms are paving the way for potential treatment opportunities targeting different resistance pathways. There is a need to characterize and define groups of patients who would benefit from PARPi rechallenge, combination therapy with PARPi, or alternative therapies. Retrospective data suggest that outcomes following PARPi are related to the prior response to platinum-based chemotherapy. Moubarak and colleagues analyzed 29 patients who had responded to the last platinum-based chemotherapy and were retreated with PARPi. The study suggested a benefit in patients with unequivocal responses to the last platinum-based chemotherapy. 15 Gadducci and colleagues analyzed the response to chemotherapy of 103 patients who received prior maintenance therapy with a PARPi and progressed. Better outcomes were seen in patients with a platinum-free interval (PFI) greater than 12 months. 16 PARPis re-exposure was prospectively investigated for the first time in the phase IIIb OReO/ ENGOT Ov-38 trial (NCT03106987). This study included women with recurrent platinumsensitive EOC who must had have responded to their most recent platinum regimen and received a prior course of maintenance PARPi. Two cohorts were enrolled, a BRCA-mutation and a non-BRCA-mutation cohort, both of which were randomized to receive olaparib or placebo until disease progression. The majority of patients had at least three lines of chemotherapy, indicating a very sensitive selected population. Rechallenge with olaparib compared to placebo modestly prolonged median PFS compared to placebo both in BRCA-mutated ( Since PARPi therapies have moved to first-line maintenance, it is important to underscore that prior PARPi exposure will not be a synonym for PARPi resistance, and further characterization will be needed. Defining the mechanisms of PARPis resistance to develop treatment strategies is an urgent unmet need. This review aims to summarize the main mechanisms of PARPi resistance described and discuss potential strategies to prevent, overcome, or delay acquired resistance to these agents.

DDR and PARP
The DDR system is essential to cell survival. PARP plays multiple roles in several DNA repair pathways, all of which could be involved in PARPi resistance. In all, 17 PARP proteins have been identified, of which PARP1, 2, and 3 have nuclear localization and are involved in DDR. PARP1 is the primary target of PARPi and is responsible for about 80-90% of the PARylation. 30 PARP1 inhibition forces cancer cells to rely on error-prone DNA repair pathways or otherwise, unrepaired damage persists into mitosis, leading to the rapid accumulation of mutations, genomic instability, and eventual cell death in the context of HR deficiency. 31 The primary mechanism of action of PARP inhibitors, as evidenced by the observed mechanisms of resistance, is trapping the PARP1 protein inducing replication fork collapse, and initiating replication fork protection. This trapping is mediated by steric factors, inhibition of the catabolic function, and interference with HPF1-modulated change in catalytic function. 32,33 While combination therapies that reinforce this trapping and its downstream consequences will be the main approach, there may also be other combination opportunities that exploit the other diverse roles of PARP in biology.
DDR pathways are also controlled by kinases, such as ATM, ATR, and DNA-protein kinase, that initiate repair signaling cascades. 34 Vulnerabilities in DDR pathways represent critical points of oncogenesis, opportunities to target novel therapeutics, and potential resistance mechanisms to genotoxic agents such as PARPi and platinum compounds. 35 Among all the resistance mechanisms, the most studied are the restoration of HR, replication fork stability, alterations in drug delivery, and signaling transduction pathways (see Figure 1). There are emerging techniques to identify mechanisms of PARPi resistance. CRISPR-based tools have provided significant insight into the mutational consequences of changes in PARP1, defining critical functional domains, and identified many of the genes we know can cause PARP inhibitor resistance. 44 While these CRISPR screens have identified multiple genes involved in resistance, with many subsequently being validated in clinical cohorts, there has also been substantial variability between findings from published screens. 45 These differences are likely heavily influenced by the genetic background the screen is undertaken in, which is often constrained by the experimental needs of such screens.

Mechanisms of resistance
The specificity of mechanisms to background is illustrated by EZH2 loss only causing resistance in BRCA2 mutants, and 53BP1 loss being involved in resistance in BRCA1 mutations 40 while increased activity of 53BP1 due to loss of KAT5 causes resistance in BRCA2-deficient cells. 46 The impact of background is likely to be driven by the essential roles many of these proteins play in cell survival, with the limits of any adaptive change imposed by how far various steps in the pathway are perturbed by existing mutations and cell type expression patterns. These subtleties will continue to be elucidated as the number of diverse screens continues to grow, and CRISPR methods that alter expression rather than causing complete loss of function are employed. Careful validation in clinical cohorts and clinical discovery research guided by the mechanistic knowledge generated in these highthroughput screens will continue to play an In addition, many studies exploring PARP resistance mechanisms and synthetic lethality are ongoing (see Table 1), and there are still many unanswered questions. These strategies include combining therapies to amplify PARPi effects, targeting the acquired vulnerabilities, and delaying resistance by suppressing the mutator phenotype in HR-mutated tumors. 47 The DDR-synthetic lethal concept is broader than PARPi, and the landscape of opportunity begins to be explored.

HRR-dependent mechanisms of PARPi resistance
Reversion mutations. Restoration of HRR function is a commonly acquired mechanism of platinum and PARPi resistance, occurring in about 20% of cases of EOC. 36 In the SOLO3 trial (NCT02282020) of olaparib versus single-agent chemotherapy in patients with BRCA mutant, platinum-sensitive relapsed EOC, 22% of patients had secondary BRCA reversion mutations upon progression. 48 These reversion mutations or reexpression of HRR-related genes that had been silenced through promoter hypermethylation may restore the HRR function. Reversion mutations   restore the open reading frame of the BRCA gene, remove the original deleterious mutation, and restore the expression of a functional protein.
Reversion mutations often show a microhomology signature, which suggests they resulted from the repair of DSBs via alternative error-prone repair mechanisms utilized in the setting of HR deficiency. 49 Somatic reversions have been observed in other HR pathway genes such as PALB2, RAD51C, and RAD51D and are associated with poor prognosis. 50,51 Furthermore, acquired loss of RAD51C promoter methylation leads to HR restoration and PARPi resistance. 52 Translational analysis of ARIEL4 phase III trial assessing rucaparib versus chemotherapy (weekly paclitaxel versus platinum-based chemotherapy) in relapsed EOC with a BRCA1 or BRCA2 mutation identified fewer BRCA reversion mutations in three of four cases with platinum-resistant disease treated with weekly paclitaxel. This is an intriguing observation that needs to be further investigated. 20 BRCA1 promoter alterations. BRCA1 or RAD51C gene silencing through promoter methylation has been detected in ovarian and breast tumors. 53 A potential mechanism of resistance to PARPi in these tumors involves gene re-expression. Preand post-platinum progression paired biopsies of EOC have shown that the de-silencing of BRCA1 is linked to platinum resistance. 54 As demonstrated in patient-derived xenograft (PDX) models of PARPi-resistant breast cancer, the loss of BRCA1 promoter methylation restores functional BRCA1 expression to the levels found in HR-proficient tumors. 42 In EOC, susceptibility to PARPi was subsequently found to require BRCA1 silencing by homozygous methylation of all copies present in the gene. 55  In the BRCA2 deficiency setting, preclinical findings of genome-wide CRISPR screen in BRCA2knockout HeLa cell lines suggested that increased 53BP1 binding near the DSBs leads to reduction in end-resection and subsequent PARPi resistance due to an increase in NHEJ. 46 These differences in mechanisms between BRCA1 and BRCA2-deficient cells potentially reflect the different steps in HR-dependent DSBs repair in which each BRCA protein acts. However, further studies are warranted to verify these differences.
Other mechanisms, such as decreased proteasomal degradation 63 and amplification of wild-type BRCA, are involved in restoring HR capacity, also leading to PARPi resistance. 64 HRR-independent mechanisms of resistance Restoration of replication fork stability. Replication stress results from the extremely increased cell growth and division in many tumors. Upon replication stress, replication forks stall and prolong cell-cycle arrest allowing time for DNA repair and re-entry into the cell cycle. 65 Damage to replication fork protection evokes genomic instability and tumorigenesis. 66 Treatment resistance promotes cancer cell progression through the cell cycle in the presence of DNA damage and replication stress; these include alterations in RAD51, 67  Signal transduction pathway. Deregulation of multiple signaling pathways has been reported to be associated with PARPi resistance. The kinase c-MET phosphorylates PARP1 leading to its activation and reducing the binding affinity of PARPi, which results in PARPi resistance. 80 Another mechanism is the upregulation of the ATM/ATR pathway, an essential checkpoint of the DNA damage response process due to its capacity to recruit DNA repair complexes through the phosphorylation of histone H2A. This phenomenon leads to HR restoration; thus, inhibiting this pathway is a strategy to overcome resistance. 81 In addition, PARPi treatment upregulates the PI3K/ AKT pro-survival pathway, which regulates cell growth and proliferation. 82

Strategies to prevent or overcome PARPi resistance
Many combinatorial strategies to overcome PARPi resistance are currently under development in preclinical or clinical settings (see Figure 2), such as PARPis combined with different anticancer agents. In this case, the combination might induce different mechanisms of actions of PARPis and contributes to the synergistic activity with each particular agent. 83 Newer DDR-targeting agents (e.g. ATR, CHK1, WEE1, and PKMYT1 inhibitors) have emerged as a proposed combinatorial strategy to bypass PARPi resistance. These agents often carry a higher toxicity burden, usually manageable with dose adjustments and supportive measures. 84 In general, inhibitors of ATR-CHK1-WEE1 are characterized by hematological toxicity, with anemia more prominent with ATR inhibitors and neutropenia with CHK1 and WEE1 inhibitors. However, safety data are still accumulating. Many combinations are thus far in early-phase stages, and additive toxicities and ideal doses are not robustly evaluated.  journals.sagepub.com/home/tam 11 with 22.9 weeks for gemcitabine plus berzosertib versus 14.7 weeks for gemcitabine alone. Grades 3/4 TRAEs with the combination were neutropenia (47%) and thrombocytopenia (24%). 92 Ongoing trials are evaluating the combination of ATRi with various chemotherapies (NCT04657068, NCT02264678, and NCT05147272). WEE1 kinase, a G2 cell-cycle checkpoint regulator, promotes cell-cycle arrest and enhances apoptosis in the setting of DNA damage. The WEE-1 inhibitor (WEE1i), adavosertib, has demonstrated synergy with PARPi in preclinical studies. 93,94 The randomized phase II non-comparative EFFORT trial (NCT03579316) showed the efficacy of adavosertib in PARPi-resistant EOC with an ORR of 23% with adavosertib alone and 29% in the combination of adavosertib plus olaparib arm. Frequent TRAEs included gastrointestinal effects (nausea, diarrhea), fatigue, and hematological toxicity. 95 Similarly, in a phase I trial with another WEE1i, ZN-c3 (NCT04158336), the most frequent adverse events were nausea, vomiting, diarrhea, and fatigue. In that study, two of 16 patients had partial response. 96 ZN-c3 is also being tested in combination with standard-ofcare chemotherapy in platinum-resistant HGSOC in a phase Ib trial (NCT04516447). Preliminary results showed an ORR of 30.2% among 43 patients. 97 More CDK1-selective than WEE1 inhibition is the PKMYT1 inhibition, which causes unscheduled activation of CDK1, leading to early mitosis. 98 Evidence from preclinical studies has demonstrated that the PKMYT1 inhibitor RP-6306 has higher selectivity for cyclin E1-overexpressing cells both in vitro and in vivo and synergy with gemcitabine. 99 Likewise, the synergy between PARPi and CDK4/6 inhibitors demonstrated in cancer cell lines derived from patients whose EOC shows high MYC expression and does not respond to PARPi. 100 DNA polymerase Polθ is an enzyme encoded by POLQ important for MMEJ repair. Preclinical studies showed a synthetic lethal interaction between loss of the POLQ gene and deficiencies in genes that control DSBs repair and HR, including BRCA1/2, ATM, and FANCD2. 101,102 Polθ is an emerging therapeutic target that confers synthetic lethality with defects in the 53BP1/Shieldin DNA repair complex. Two studies report specific Polθ inhibitors with in vivo efficacy, which could underpin a promising therapy to bypass PARPi resistance in HR-deficient tumors. 103,104 Next-generation PARP1-selective inhibitors have entered phase I clinical trials. Current PARPis are equipotent against PARP1 and PARP2 enzymes. Animal data suggest that these drugs exert their therapeutic effects through PARP1, and toxicity comes mainly from the inhibition of PARP2. 105 The initial data from the ongoing phase I/II PETRA trial (NCT04644068) of AZD5305, a highly selective PARP1 inhibitor, demonstrated favorable tolerability across different doses with no dose-limiting toxicities and preliminary signals of activity. The PETRA study included women with advanced EOC harboring a germline or somatic BRCA1/2, PALB2, or RAD51C/D mutation, who had received prior platinum and prior PARPi. 106 AZD5305 is also being tested in combination with chemotherapy and antibody-drug conjugates (ADC) targeting HER2 or topoisomerase 1 (NCT04644068).

Indirect inhibition of HR
Target oncoproteins such as EGFR, VEGF, MEK, and PI3K-AKT have been reported to impair HR indirectly. 50 In a phase I trial combining olaparib with AKT inhibitor capicarsetib, 11 of 25 patients with EOC achieved clinical benefit, irrespective of BRCA1/2 mutation. 107 PARPi resistance is associated with the upregulation of the RAS/MAPK pathway. Therefore, MEK inhibition induced HR deficiency by decreasing MRE11, RAD50, NBN, and BRCA1/2. The ongoing phase I/II SOLAR trial (NCT03162627) combines MEK inhibitor selumetinib with olaparib and includes an expansion cohort of PARPiresistant EOC. Preclinically, these two agents synergistically act to increase DNA damage and apoptosis in response to PARPi. 108 Another way to inhibit HR indirectly is by targeting epigenetic regulators. Bromodomain containing 4 (BRD4) is a member of the BET protein family with roles in epigenetic gene regulation. BRD4 inhibitors have been shown to suppress HR-associated genes, including CTIP, BRCA1, RAD51, and TOPBP1, thereby generating a state of HR deficiency and synergy with PARPi. 109,110 BET and PARP inhibition have demonstrated synergy in xenograft models with a reduction in tumor growth, increasing apoptosis, and DNA damage. 111 Preclinically, BET inhibition has been shown to enhance antitumor immunity. 112

Modulation of the tumor microenvironment
The tumor microenvironment protects tumor cells by providing mechanical support or secreting a range of cytokines, enabling cancer to evade both the immune system and subsequent therapies. Cancer-associated fibroblasts (CAFs) participate in anticancer drug resistance by upregulating desmoplasia and pro-survival mechanisms within the tumor microenvironment. 113 Tumor stroma is a complex component of the tumor microenvironment that contains numerous CAFs. Cytokine secretion, excess deposition, and aberrant remodeling of the extracellular matrix allow tumor cells to proliferate rapidly, develop resistance to therapy, and escape from immune surveillance. 77 A high stromal cell ratio and extensive stromal desmoplasia have been reported as features of acquired chemo-resistance in the HGSOC. 54 Strategies that modulate the immune microenvironment may overcome PARPi resistance in EOC.
Altered angiogenesis, desmoplastic stroma, and upregulation of drug efflux pump all impair drug delivery to tumor cells. ADC is a complex emerging therapy that consists of an antibody designed against a specific tumor cell target conjugated by a linker to a cytotoxic payload. 114 Targeting tumor cell surface antigens and delivering the payload may be effective ways to overcome drug resistance. The single-arm phase III SORAYA trial evaluated Mirvetuximab soravtansine in 106 women with platinum-resistant HGSOC and high expression of folate receptor alpha. Patients were heavily pretreated, and 48% had received prior PARPi. The ORR was 32.4%, including five complete responses. 115 In the post-PARP setting, the most critical factor in developing ADCs is to find relevant antigens to target in this population with a high affinity to ensure appropriate drug release into the cancer cell.
Antiangiogenic therapy induces a hypoxemic tumor microenvironment with the downregulation of HR genes and can potentially improve drug delivery. However, the phase IIb CONCERTO trial (NCT02889900) demonstrated low clinical activity of the cediranibolaparib combination in heavily pretreated patients with BRCA wild-type and platinumresistant EOC with ORR of 15.3% (9/59 patients). 116 TRAEs were experienced by 40% of patients. The most common TRAEs were hypertension, fatigue, diarrhea, and nausea. 116 The phase II EVOLVE trial assessed this combination of cediranib-olaparib in patients after progression on PARPi. 51 Patients were enrolled into platinum-sensitive (n = 10), platinum-resistant (n = 10), or exploratory (n = 10) cohort of patients who had progressed on a PARPi and progressed again on subsequent standard chemotherapy, regardless of platinum sensitivity. The 16-week PFS was 55%, 50%, and 39%, respectively. The ORR was 20% in the platinum-resistant cohort as opposed to 0% in platinum-sensitive and 8% in exploratory cohorts. Grade 3 TRAEs were reported in 38% of patients, most commonly diarrhea and anemia. 51 Translational analyses of EVOLVE with paired biopsies identified acquired mechanisms of PARP resistance, regardless of the platinum cohort. Patients with reversion mutations in BRCA1/2 and other HR genes and ABCB1 upregulation had worse outcomes than patients with intact BRCA1/2 genes. The rate of cyclin E1 (CCNE1) amplification, a biomarker of platinum resistance, was higher in BRCA1/2-mutated than in BRCA1/2 wild-type (33% versus 15%) tumors. This result was the opposite of prior reports 117 and could be driven by heavily pretreated patients with partial active synthetic lethality. Downregulation of SLFN11 was found in 7% of patients. The data suggest that patients with reversion mutations and ABCB1 upregulation might not have benefited from the combination of cediranib and olaparib. 51 Ongoing NIRVANA-R phase II trial is assessing niraparib and bevacizumab in patients with platinum-sensitive recurrence previously treated with PARPi (NCT04734665).
The combination of the DDR pathway and immune checkpoint inhibitors is an area of active investigation. PARPi propagates DNA damage and releases DNA fragments into the cytoplasm, activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. This pathway recognizes extranuclear double-stranded DNA and triggers the IRF3-type I interferon pathway, which is a crucial mediator of the immune system and induces activation of different immune cell types. 118,119 PARP inhibition has also been shown to inactivate GSK3β and increase the CD8 + T-cell infiltration. 120 However, there is a debate if these pathways depending on the BRCA mutation status and may AC Veneziani, C Scott et al.
journals.sagepub.com/home/tam 13 be compromised if the mechanism of PARP resistance is the restoration of HR proficiency. 121,122 Another study with cell lines has demonstrated that the activation of the cGAS-STING pathway occurs either solely or more potently in BRCA-deficient tumors. 123 However, data from the phase II trial (NCT02484404) combining olaparib and durvalumab did not support the preclinical findings of modulation of cGAS/STING pathway by PARPi. In this study, STING expression was not associated with clinical benefit. 124 Other combinations of PARPi and immune checkpoint inhibitors in recurrent EOC were assessed in small studies and were generally well tolerated. 125 The phase I/II basket trial MEDIOLA (NCT02734004) included one cohort of platinum-sensitive EOC with germline BRCA1/2 mutation and previously treated with at least one platinum-based chemotherapy. 126 The combination of durvalumab and olaparib demonstrated an ORR of 71.9% in this cohort. A second-stage phase II study was designed to test the addition of bevacizumab to olaparib and durvalumab in germline BRCA wild-type relapsed EOC patients. 127 The triplet demonstrated superiority over the doublet for all the endpoints with an ORR of 87.1% (95% CI: 70.2-96.4) versus 34.4% (95% CI: 18.6-53.2). 127 Recent data confirmed the efficacy with median OS of 26.1 months for olaparib plus durvalumab and 31.9 months for olaparib, durvalumab, and bevacizumab. The most common TRAEs were anemia and hypertension in patients who received the triplet combination. 128 The phase I/II TOPACIO trial (NCT02657889) assessed the combination of pembrolizumab and niraparib 129 in platinum-resistant EOC regardless of BRCA status. The ORR of 18% with the combination was reported; no significant differences were seen between patients with BRCA1/2 mutation and wild-type tumors. TRAEs of at least grade 3 included anemia (11%), thrombocytopenia (9%), and hyperglycemia (4%). 129 A phase II proof-ofconcept trial assessing the combination of durvalumab and olaparib demonstrated an ORR of 14% (5/35 patients) in a predominantly platinumresistant recurrent EOC. This study indicated immunomodulatory effects of olaparib/durvalumab in patients and that VEGF/VEGFR pathway blockade would improve the tumor responses. 124 Recently, MOONSTONE/GOG-3032 phase II trial (NCT03955471) evaluated the combination of niraparib and dostarlimab in patients with platinum-resistant EOC without BRCA mutation who received prior bevacizumab. The ORR was 7.3% (3/41 patients), and the median PFS was 2.1 months. Futility was declared due to low ORR. 130 Clinical trials in first-line evaluating the combination of a PARPi with antiangiogenics or programmed cell death protein 1/programmed cell death ligand 1 inhibitors are ongoing, and results are awaited (NCT03602859, NCT037401165, NCT03737643, NCT03522246).

Biomarkers of resistance to PARPi
A wide range of mechanisms result in PARP resistance, and each individual with EOC may have more than one altered pathway. Furthermore, all mechanisms have the potential to evolve, and predicting PARPi resistance in the clinic is challenging. Platinum sensitivity strongly predicts response to PARPi, and their resistance mechanisms often overlap. There is no biological predictive biomarker of platinum resistance, and PFI is widely used as a clinical biomarker. However, this definition has been questioned and replaced by platinum treatment-free interval (TFIp), considered a continuous variable. HRD is a dynamic phenotype useful to predict response to platinum agents and PARPi. 131 Clinically, HRD test results and PARPi responses can be discordant. This may be because tumors with reversion mutations remain with evidence of HRD on these tests or that an alternative HR-independent mechanism of resistance is prevailing. 132 Therefore, methods to reliably determine the HRD status in HGSOC are of critical importance to stratify and optimize treatment. There are three main categories of HRD tests: HRR pathway-related genes, genomic scars or mutational signatures, and functional assays. 133 Currently, the most common commercial tests are the myChoice CDx (Myriad Genetics) and Foundation Focus CDx BRCA LOH (Foundation Medicine). The myChoice CDx use next-generation sequencing to assess tumor Genomic Instability Score (GIS), which is a combination of copy number variations measures, including loss of heterozygosis (LOH), telomeric allelic imbalance, and large-scale state transitions. The Foundation Focus CDx BRCA LOH assesses large-scale LOH at the genomic level. Both assays include a BRCA mutation test. GIS is a continuous score that varies between 0 and 100. A tumor is considered 'HRD positive' when GIS ⩾ 42. The percentage of genomic LOH considered TherapeuTic advances in Medical Oncology Volume 15 14 journals.sagepub.com/home/tam 'HRD positive' was 14% in the ARIEL2 trial 134 and 16% in the ARIEL3 trial. 135 Other non-commercial HRD tests are under development, such as the assay of KU Leuven and collaborators, which was recently presented in comparison with Myriad myChoice in patients from the PAOLA-1 trial. The Leuven HRD test showed a similar impact of olaparib on median PFS as Myriad myChoice test. 136 Other integrative models based on mutational signatures have been developed. HRDetect 137 and Classifier of Homologous Recombination Deficiency (CHORD) 138 are HRD tests based on whole-genome sequencing (WGS) analysis. These tests reflect the accumulation of genomic mutational scars and correlate with HRD in BRCA-deficient tumors. CHORD was developed using WGS data of 3584 patients from a pan-cancer metastatic cohort, including EOC. CHORD uses a combination of 29 mutational features of three somatic mutation categories: single-base substitution, insertions and deletions, and structural variants. 138 CHORD could detect HRD with overall low false-positive (<2%) and falsenegative rates (<6%). 138 HRDetect was first tested for breast cancer and combines HRDinduced point mutations and short indels with large-scale chromosomal alterations. HRDetect for EOC performed similarly well compared to HRD score by Myriad myChoice or Foundation Focus LOH tests. 139 However, all tests that are based on assessing genomic scars have limitations and still miss many potential responders to DNAdamaging therapies or falsely predict a benefit from DNA-damaging therapies in situations where HR may have been restored.
Functional assays assessing current HRD status are under investigation and require validation before clinical use. 140 Recently, the RECAP (Repair CAPacity) assay in breast cancer was published. The RECAP test is based on measuring RAD51 foci formation in proliferating cells by immunofluorescence, and its concordance with HRDetect and CHORD was 70%. 141 The basal RAD51 foci score based on HR status, BRCA1 promoter methylation, and the HRDetect score showed a correlation with PARPi activity in EOC PDXs. The expression level of RAD51 foci was strongly inversely correlated with olaparib responsiveness. In this study, the lower the foci score, the greater the sensitivity to olaparib. 142  The development of drugs that target the DNA replication process is increasing the interest in biomarkers of replication stress. Konstatinopoulos and colleagues analyzed the replication stress biomarkers 150 in patients from the phase II trial with gemcitabine alone or in combination with berzosertib (ATRi) (NCT02595892). 92 Replication stress high tumors were defined as having at least one genomic replication stress alteration: loss of retinoblastoma pathway regulation (CCNE1 amplification, RB1 two-copy loss, CDKN2A twocopy loss), and/or oncogene-induced replication stress (KRAS amplification, NF1 mutations, ERBB2 amplification, MYC amplification, and MYCL1 amplification). Patients with high replication stress tumors had prolonged PFS on gemcitabine monotherapy. 150 Gemcitabine itself induces replication stress, and in cells already under stress, it provokes cell death. 151 On the other hand, patients with low replication stress responded poorly to gemcitabine alone and benefited from the addition of the ATRi berzosertib, 150 which may explain the synergism between them. Further investigations are needed to define replication stress and how to identify patients in the clinic.

Conclusion
PARPi resistance is emerging as a common challenge in the clinic and will increase with the wide use of PARPi in the first-line setting, despite long treatment-free intervals and likely cure for more women. Exploiting additional DDR targets is a promising strategy for PARPi combination or sequential therapies. The primary issue will be the selection of patients, given the heterogeneity of both HGSOCs and PARPi mechanisms of resistance. Therefore, efforts must be made to identify and integrate biomarkers to target tumor molecular vulnerabilities. Biomarkers of PARPi resistance need to be validated in further studies, considering feasibility, cost, and applicability to clinical practice. Functional assays are under investigation and may provide more accurate HRD status and prediction of PARPi response. New therapeutic targets extending the concept of synthetic lethality provide an exciting area of research to overcome PARPi resistance. Nonetheless, much coordinated preclinical, clinical, and translational work will be necessary to bring these advances to clinical practice.

Declarations
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